Ecl plus chemiluminescent substrate

Tissues were minced and sonicated in RIPA lysis buffer containing protease and phosphatase inhibitors (Pierce). Protein concentrations were determined using the bicinchoninic acid colorimetric method (Pierce). Tumor lysates were diluted in reducing sample buffer (Novex) and 50 μg were loaded per lane on a 4% to 12% SDS-PAGE gel (Nu-Page) and run at 160 volts (0.8 volt hours). Gels were then transferred to nitrocellulose at 7 volts (BioRad), blocked using 5% non-fat dry milk/0.05 mM tris buffered saline with 0.05% tween-20 for 1 hr, incubated at 1:1000 in anti-OX2, 200 μg/ml (Santa Cruz) in blocking buffer for 1 hr, and washed six times over 1 hr in TBS/Tween-20. Blots were then incubated at 1:10,000 with anti-goat, 500 μg/0.5 ml IgG HRP (Jackson ImmunoResearch) in blocking buffer for 1 hour and washed six times over 1 hour in TBS/Tween-20. nitrocellulose was incubated in ECL Plus chemiluminescent substrate (GE) for 1 minute and exposed to HyBlot CL Autoradiography film (Denville Scientific) for 30 seconds.

The Western blot technique was applied as described previously [25] (link). Briefly, stationary phase promastigotes of L infantum H-line or wild-type parasite (1×10⁷ cells per lane) were washed with ice-cold PBS three times, and disrupted by sonication. An equal volume of sample buffer [0.1 M Tris (Merck), 12% sodium dodecyl sulfate (Merck), 10% glycerol (Merck), 5% β-mercaptoethanol (Merck), 0.1% bromophenol blue, pH 8.0] was mixed and the solution denatured at 95°C for 5 min. Promastigote lysates were fractioned individually on a 12% SDS-PAGE gel and subsequently transferred onto a nitrocellulose membrane (Sigma-Aldrich). The blots were individually incubated with 1∶50 diluted sera in PBS containing 3% skimmed milk at room temperature for 18 h. The blots were incubated with 1∶10000 diluted goat anti-dog IgG-heavy and light chains antibody horseradish peroxidase (HRP) conjugated (Bethyl Lab. Inc) in PBS containing 5% skimmed milk at room for 2 h. The blots were washed as above, incubated with ECL Plus chemiluminescent substrate (GE Healthcare), and exposed to X-ray film.